Low sensitivity of acid-fast bacilli (AFB) sputum smears and absence of productive cough are obstacles to the diagnosis of pulmonary tuberculosis (PTB) in hospitals that lack access to bronchoscopy. To evaluate induced sputum, gastric content, blood and urine specimens to improve PTB diagnosis in patients not diagnosed by expectorated sputum AFB smears. Patients admitted to the medical wards of a large public hospital in Gaborone, Botswana, were prospectively enrolled if they had symptoms consistent with PTB, an abnormal chest radiograph, were treated empirically with anti-tuberculosis chemotherapy or had no improvement on antibiotics, and had a non-productive cough or AFB smear-negative sputum. Induced sputum was stained for AFB and Mycobacterium tuberculosis cultures were performed on induced sputum, gastric contents, urine and blood. Of 140 patients meeting the enrollment criteria, 113 (81%) were human immunodeficiency virus (HIV) positive. Fifty-seven (41%) had PTB based on positive cultures from one or more sites, including 48 (84%) from induced sputum, 17 (30%) urine, 13 (23%) gastric contents and 7 (12%) blood. AFB smears were positive in only 18 (32%) culture-proven PTB cases. Induced sputum cultures greatly enhanced M. tuberculosis detection in patients with a high prevalence of HIV/AIDS in a hospital without access to bronchoscopy.